Methods of producing stable pancreatic enzyme compositions

ABSTRACT

Compositions of the present invention, comprising at least one digestive enzyme (e.g., pancrelipase) are useful for treating or preventing disorders associated with digestive enzyme deficiencies. The compositions of the present invention can comprise a plurality of coated particles, each of which is comprised of a core coated with an enteric coating comprising at least one enteric polymer and 4-10% of at least one alkalinizing agent, or have moisture contents of about 3% or less, water activities of about 0.6 or less, or exhibit a loss of activity of no more than about 15% after six months of accelerated stability testing.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims priority to U.S. Provisional ApplicationNo. 60/902,091 filed Feb. 20, 2007, U.S. Provisional Application No.60/902,093 filed Feb. 20, 2007, and U.S. Provisional Application No.60/902,092 filed Feb. 20, 2007, the disclosures of which are each hereinincorporated by reference in their entirety for all purposes.

BACKGROUND OF THE INVENTION

In cases of pancreatic insufficiency, pancrelipase and other pancreaticenzymes products (PEPs) can be administered to at least partially remedythe enzyme deficiency caused by various diseases affecting the pancreas,such as pancreatitis, pancreatectomy, cystic fibrosis, etc. The use ofpancreatic enzymes in the treatment of pancreatic insufficiency is anessential part of the therapy of patients afflicted with cysticfibrosis. Without these supplements, patients become severelynutritionally impaired. This nutritional impairment can be lifethreatening if left untreated, particularly in the case of infants.

The drug substance pancrelipase is mainly a combination of three enzymeclasses: lipase, protease and amylase, together with their variousco-factors and co-enzymes. These enzymes are produced naturally in thepancreas and are important in the digestion of fats, proteins andcarbohydrates. Pancrelipase is typically prepared from porcinepancreatic glands, although other sources can also be used, for examplethose described in U.S. Pat. No. 6,051,220, U.S. 2004/0057944,2001/0046493, and WO 2006044529, each of which is herein incorporated byreference. The enzymes catalyze the hydrolysis of fats into glycerol andfatty acids, starch into dextrin and sugars, and protein into aminoacids and derived substances.

Pancreatic enzymes show optimal activity under near neutral and slightlyalkaline conditions. Under gastric conditions, pancreatic enzymes may beinactivated with a resulting loss in biological activity. Therefore,exogenously administered enzymes are generally protected against gastricinactivation and remain intact during their transit through the stomachand into the duodenum. Although it is desirable to coat pancreaticenzymes, uncoated preparations are also found in commerce. Pancreaticlipases are the most sensitive to gastric inactivation and are the mostimportant single enzymes in the treatment of malabsorption. Lipaseactivity is typically monitored to determine the stability of an enzymecomposition containing lipase.

After passage through the stomach, the enzymes should be released in theduodenum within 5-30 minutes, since digestion by pancreatic enzymes andabsorption of the metabolites takes place primarily in the upper segmentof the intestine, although digestion and absorption can take placethroughout GI transit. Pancreatic enzymes have typically been coatedwith an enteric coating polymer, which protects the enzyme compositionagainst the acidic environment of the stomach and then provides releaseof the enzyme in the intestine.

The conventional pancreatic enzyme preparations are intrinsicallyunstable and do not possess the shelf-life typically associated withapproved pharmaceutical products for oral use. The activity ofpancreatic enzyme preparations is typically determined based on theactivity of lipase, which is one of the enzymes most sensitive to losingenzymatic activity during storage. Commercially available digestiveenzyme compositions show a loss of lipase activity over time of up toabout 35% or more. In order to compensate for the losses of enzymaticactivity during storage and to ensure that the product provides thelabel-claimed potency at the end of the shelf life, manufacturerstypically overfill the dosage forms from 5% to 60% and current USPspecifications for Pancrelipase Delayed-Release Capsules allow forPancrelipase equivalent to not less than 90% and not more than 165% ofthe labeled lipase activity.

In practice this means that patients and prescribers are unable to judgethe dosage strength with accuracy, with the practical result that theappropriate dosage needs to be determined empirically for each newprescription. Patients with exocrine pancreatic insufficiency disordersrely on these drugs to provide the enzymes they need to digest foodproperly. If the label contains an inaccurate statement about aparticular product's potency, then the patient is at risk for receivingtoo much or too little of the medicine.

Accordingly, it would be desirable to provide a stable digestive enzymecomposition capable of maintaining the necessary activity for theexpected shelf life of the enzyme preparation, without depending onoverfilling the dosage form.

SUMMARY OF THE INVENTION

The present invention relates to stable digestive enzyme compositionsand dosage forms and methods for producing stable enzyme compositionsand dosage forms. More particularly, the present invention relates toenteric coated enzyme compositions and dosage forms that exhibit minimalloss of activity under typical storage conditions.

In one embodiment, the present invention provides a compositioncomprising at least one digestive enzyme, wherein the composition has amoisture content of about 3% or less.

In another embodiment, a composition of the present invention comprisesat least one digestive enzyme, wherein the composition has a wateractivity of about 0.6 or less.

In another embodiment, a composition of the present invention comprisesat least one stabilized digestive enzyme, wherein the at least onestabilized digestive enzyme exhibits a loss of activity of no more thanabout 15% after six months of accelerated stability testing.

In yet another embodiment, the present invention provides a dosage formsuch as a tablet or a capsule filled with the composition of the presentinvention.

In yet another embodiment, a composition of the present inventionfurther comprises the at least one digestive enzyme coated with acoating, wherein the coating comprises an enteric polymer and optionallyat least one inorganic material.

In yet another embodiment, the present invention provides a packagecomprising a sealed container made of moisture resistant material, adesiccant, and at least one dosage form of the present invention,wherein the desiccant and at least one dosage form are inside the sealedcontainer.

In yet another embodiment, the present invention provides a method oftreating or preventing a disorder associated with digestive enzymedeficiency comprising administering a composition of the presentinvention to a mammal in need thereof.

In yet another embodiment, the present invention provides a method ofpreparing a composition of the present invention. In one embodiment, themethod comprises coating particles of the at least one digestive enzymein an atmosphere having a moisture content of about 3.6 g water per m³or less, with a coating comprising an enteric polymer and at least oneinorganic material, thereby forming a plurality of delayed releaseparticles.

DETAILED DESCRIPTION OF THE INVENTION

One aspect of the present invention is directed to a stabilizeddigestive enzyme composition. The term “stabilized digestive enzyme”means a digestive enzyme which maintains substantial enzymatic activityafter long-term storage. The term “digestive enzyme” denotes an enzymein the alimentary tract which breaks down the components of food so thatthey can be taken or absorbed by the organism.

Non-limiting classes of digestive enzymes suitable for use in thepresent invention include lipases, amylases and proteases. Non-limitingexamples of digestive enzymes include pancrelipase (also referred to aspancreatin), lipase, co-lipase, trypsin, chymotrypsin, chymotrypsin B,pancreatopeptidase, carboxypeptidase A, carboxypeptidase B, glycerolester hydrolase, phospholipase, sterol ester hydrolase, elastase,kininogenase, ribonuclease, deoxyribonuclease, α-amylase, papain,chymopapain, glutenase, bromelain, ficin, β-amylase, cellulase,β-Galactosidase, lactase, sucrase, isomaltase, and mixtures thereof.

In one embodiment of the present invention, the stabilized digestiveenzyme is a pancreatic enzyme. The term “pancreatic enzyme” as usedherein refers to any one of the enzyme types present in the pancreaticsecretion, such as amylase, lipase, protease, or mixtures thereof, orany extractive of pancreatic origin having enzymatic activity, such aspancreatin. The pancreatic enzyme can be obtained through extractionfrom the pancreas, produced artificially, or obtained from sources otherthan the pancreas, such as from microbes, plants or other animaltissues.

In another embodiment of the present invention, the stabilized digestiveenzyme is pancrelipase. The terms “pancrelipase” or “pancreatin” denotea mixture of several types of enzymes, including amylase, lipase, andprotease enzymes. Pancrelipase is commercially available, for examplefrom Nordmark Arzneimittel GmbH, or Scientific Protein Laboratories LLC.

In one embodiment of the compositions of the present invention, thestabilized digestive enzyme comprises a lipase. The term “lipase” refersto an enzyme that catalyzes the hydrolysis of lipids to glycerol andsimple fatty acids.

Examples of lipases suitable for the present invention include, but arenot limited to animal lipase (e.g., porcine lipase), bacterial lipase(e.g., Pseudomonas lipase and/or Burkholderia lipase), fungal lipase,plant lipase, recombinant lipase (e.g., produced via recombinant DNAtechnology by a suitable host cell, selected from any one of bacteria,yeast, fungi, plant, insect or mammalian host cells in culture, orrecombinant lipases which include an amino acid sequence that ishomologous or substantially identical to a naturally occurring sequence,lipases encoded by a nucleic acid that is homologous or substantiallyidentical to a naturally occurring lipase-encoding nucleic acid, etc.),chemically-modified lipase, or mixtures thereof.

In another embodiment of the compositions of the present invention, thestabilized digestive enzyme comprises an amylase. The term “amylase”refers to glycoside hydrolase enzymes that break down starch, forexample α-amylases, β-amylases, γ-amylases, acid α-glucosidases,salivary amylases such as ptyalin, etc.

The amylases suitable for use in the compositions of the presentinvention include, but are not limited to animal amylases, bacterialamylases, fungal amylases (e.g., Aspergillus amylase and, preferably, isAspergillus oryzae amylase), plant amylases, recombinant amylases (e.g.,produced via recombinant DNA technology by a suitable host cell,selected from any one of bacteria, yeast, fungi, plant, insect ormammalian host cells in culture, or recombinant amylases which includean amino acid sequence that is homologous or substantially identical toa naturally occurring sequence, amylases encoded by a nucleic acid thatis homologous or substantially identical to a naturally occurringamylase-encoding nucleic acid, etc.), chemically modified amylases, ormixtures thereof.

In another embodiment of the compositions of the present invention, thestabilized digestive enzyme comprises a protease. The term “protease”refers generally to enzymes (e.g., proteinases, peptidases, orproteolytic enzymes) that break peptide bonds between amino acids ofproteins. Proteases are generally identified by their catalytic type,e.g., aspartic acid peptidases, cysteine (thiol) peptidases,metallopeptidases, serine peptidases, threonine peptidases, alkaline orsemi-alkaline proteases, neutral and peptidases of unknown catalyticmechanism.

Non-limiting examples of proteases suitable for use in the compositionsor oral dosage forms of the present invention include serine proteases,threonine proteases, cysteine proteases, aspartic acid proteases (e.g.,plasmepsin) metalloproteases, glutamic acid proteases, etc. in addition,proteases suitable for use in the compositions or oral dosage forms ofthe present invention include, but are not limited to animal proteases,bacterial proteases, fungal proteases (e.g., an Aspergillus melleusprotease), plant proteases, recombinant proteases (e.g., produced viarecombinant DNA technology by a suitable host cell, selected from anyone of bacteria, yeast, fungi, plant, insect or mammalian host cells inculture, or recombinant proteases which include an amino acid sequencethat is homologous or substantially identical to a naturally occurringsequence, proteases encoded by a nucleic acid that is homologous orsubstantially identical to a naturally occurring protease-encodingnucleic acid, etc.), chemically modified proteases, or mixtures thereof.

The compositions or oral dosage forms of the present invention cancomprise one or more lipases (i.e., one lipase, or two or more lipases),one or more amylases (i.e., one amylase, or two or more amylases), oneor more proteases (i.e., one protease, or two or more proteases),mixtures of one or more lipases with one or more amylases, mixtures ofone or more lipases with one or more proteases, mixtures of one or moreamylases with one or more proteases, or mixtures of one or more lipaseswith one or more amylases and one or more proteases.

In one embodiment, the digestive enzyme is a porcine pancreatic extractcomprising various lipases (e.g., lipase, colipase, phospholipase A2,cholesterol esterase), proteases (e.g., trypsin, chymotrypsin,carboxypeptidase A and B, elastase, kininogenase, trypsin inhibitor),amylases, and optionally nucleases (ribonuclease, deoxyribonuclease). Inanother embodiment, the digestive enzyme is substantially similar tohuman pancreatic fluid. In yet another embodiment, the digestive enzymeis pancrelipase USP. In still another embodiment, the digestive enzymeis pancrelipase ESP having a lipase activity of 69-120 U USP/mg, amylaseactivity of greater than or equal to 216 U USP/mg, protease activity ofgreater than or equal to 264 U USP/mg, and total protease activity ofgreater than or equal to 264 U USP/mg.

Lipase activities in the compositions or oral dosage forms of thepresent invention can be about 4500-25,000 IU, for example about4500-5500 IU, about 9000-11,000 IU, about 13,500-16,500 IU, and about18,000-22,000 IU. Amylase activities in the compositions or oral dosageforms of the present invention can be about 8100-180,000 IU, for exampleabout 8000-45,000 IU, about 17,000-90,000 IU, about 26,000-135,000 IU,about 35,000-180,000 IU. Protease activities in the compositions or oraldosage forms of the present invention can be about 8000-134,000 IU, forexample about 8000-34,000 IU, 17,000-67,000 IU, 26,000-100,000 IU,35,000-134,000 IU. In one embodiment, the lipase activity ranges fromabout 4500-5500 IU, the amylase activity ranges from about 8000-45,000IU, and the protease activity ranges from about 8000-34,000 IU. Inanother embodiment, the lipase activity ranges from about 9000-11,000IU, the amylase activity ranges from about 17,000-90,000 IU, and theprotease activity ranges from about 17,000-67,000 IU. In yet anotherembodiment, the lipase activity ranges from about 13,500-16,500 IU, theamylase activity ranges from about 26,000-135,000 IU, and the proteaseactivity ranges from about 26,000-100,000 IU. In still anotherembodiment, the lipase activity ranges from about 18,000-22,000 IU, theamylase activity ranges from about 35,000-180,000 IU, and the proteaseactivity ranges from about 35,000-134,000 IU.

The ratios of lipase:protease:amylase in the compositions or oral dosageforms of the present invention can be in the range of about 1:10:10 toabout 10:1:1, or about 1.0:1.0:0.15 (based on enzyme activities). Theratio of amylase/lipase in the compositions or oral dosage forms of thepresent invention can range from about 1.8-8.2, for example about1.9-8.2, and about 2.0-8.2. The ratio of protease/lipase in thecompositions or oral dosage forms of the present invention can rangefrom about 1.8-6.2, for example about 1.9-6.1, and about 2.0-6.1.

In another embodiment, the activities of lipase, protease, and amylasecan be those described in Table A, below:

TABLE A Formulation 1 2 3 4 min max min max min max min max Activity(IU) Lipase 4500 5500 9000 11000 13500 16500 18000 22000 Amylase 810045000 17100 90000 26100 135000 35100 180000 Protease 8100 34000 1710067000 26100 100000 35100 134000 Ratio Amylase/Lipase 1.8 8.2 1.9 8.2 1.98.2 2.0 8.2 Protease/Lipase 1.8 6.2 1.9 6.1 1.9 6.1 2.0 6.1

The total amount of digestive enzymes (by weight) in the compositions ororal dosage forms of the present invention can be about 20-100%, 20-90%,20-80%, 20-70%, 20-60%, 20-50%, 20-40%, 20-30%, or about 20%, about 30%,about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, orabout 100%. In one embodiment, the total amount of digestive enzymes is60-90%. In another embodiment, the total amount of digestive enzymes(e.g., pancrelipase) is about 68-72%.

In one embodiment, the compositions or oral dosage forms of the presentinvention, comprising at least one digestive enzyme, have a moisturecontent of about 3% or less, about 2.5% or less, about 2% or less, about1.5% or less, or about 1% or less, inclusive of all ranges and subrangestherebetween (i.e., any of about 2.5% to 3%, 2% to 3%, 1.5% to 3%, 1% to3%, 2% to 2.5%, 1.5% to 2.5%, 1% to 2.5%, 1.5% to 2%, 1% to 2%, 1% to1.5%, etc.). Compositions or oral dosage forms of the present invention,maintained at low moisture content, have been found to be substantiallymore stable compared to conventional compositions maintained at highermoisture contents, e.g. above about 3% or higher.

The term “moisture content”, also referred to as “water content”, meansthe amount of water that a composition contains. For compositions whichdo not change volume with changing moisture content, the moisturecontent can be expressed volumetrically (i.e., by volume) as the ratioof the mass of moisture to the dry volume of the material. Forcompositions that change volume with changing moisture content, themoisture content can be expressed gravimetrically (i.e., by weight) asthe mass of water removed upon drying per unit dry mass of the specimen.Determination of moisture content can be achieved by any of theconventional methods known in the art. For example, the moisture contentcan be determined by chemical titration, such as Karl Fischer titration,in which a sample is dissolved in an electrochemical titration cell.Water from the sample is consumed in an electrochemical reaction whoseendpoint is measured potentiometrically, thereby providing a directmeasure of the amount of water in the sample. Alternatively, relativelysimple thermogravimetric methods may be used such as “Loss on Drying”(LoD), in which the mass of a sample is measured prior to, and aftercontrolled drying. The loss of mass after drying is attributed to lossof moisture. Commercially available moisture analyzers (e.g., availablefrom Mettler Toledo, Sartorius AG, etc.) can also be used to determinemoisture content. The moisture content of the compositions or oraldosage forms of the present invention can be measured by any suitablemethod known in the art, for example LoD.

In another embodiment, the compositions or oral dosage forms of thepresent invention, comprising at least one digestive enzyme, have awater activity of about 0.6 or less, about 0.5 or less, about 0.4 orless, about 0.3 or less, about 0.2 or less, or about 0.1 or less,inclusive of all ranges and subranges therebetween (i.e., any of about0.5 to 0.6, 0.4 to 0.6, 0.3 to 0.6, 0.2 to 0.6, 0.1 to 0.6, 0.4 to 0.5,0.3 to 0.5, 0.2 to 0.5, 0.1 to 0.5, 0.3 to 0.4, 0.2 to 0.4, 0.1 to 0.4,0.2 to 0.3, 0.1 to 0.3, 0.1 to 0.2, etc.). Compositions or oral dosageforms of the present invention, maintained at a low water activity, havebeen found to be substantially more stable compared to conventionaldigestive enzyme compositions maintained at higher water activitylevels.

Water activity, also referred to as “aw”, is the relative availabilityof water in a substance. As used herein, the term “water activity” isdefined as the vapor pressure of water in a sample divided by the vaporpressure of pure water at the same temperature. Pure distilled water hasa water activity of exactly one. Water activity is temperaturedependent. That is, water activity changes as the temperature changes.In the present invention, water activity is measured at a temperatureranging from about 0° C. to about 50° C., preferably from about 10° C.to about 40° C.

The water activity of a product can be determined by measuring therelative humidity of the air surrounding the sample at equilibrium.Accordingly, measurement of water activity in a sample is typicallycarried out in an enclosed (usually insulated) space where thisequilibrium can take place. At equilibrium, the water activity of thesample and the relative humidity of the air are equal, and therefore ameasurement of the equilibrium relative humidity (ERH) of the air in thechamber provides a measure of the water activity of the sample. At leasttwo different types of water activity instruments are commerciallyavailable. One type of water activity instruments uses chilled-mirrordewpoint technology (e.g., AquaLab™ water activity meters available fromDecagon Devices, Inc.) while others measure relative humidity withsensors that change electrical resistance or capacitance (e.g., wateractivity meters available from Rotronic). The water activity of thecompositions or oral dosage forms of the present invention can bemeasured by any suitable method known in the art.

In another embodiment, the compositions or oral dosage forms of thepresent invention, comprising at least one stabilized digestive enzyme,exhibit a loss of enzyme activity of no more than about 25%, no morethan about 20%, no more than about 15%, no more than about 12%, no morethan about 10%, no more than about 8%, or no more than about 5%, aftersix months of accelerated stability testing.

The term “accelerated stability testing” or “accelerated storagetesting” refers to test methods used to simulate the effects ofrelatively long-term storage conditions on enzyme activity, which can becarried out in a relatively short time. Accelerated stability testingmethods are known in the art to be a reliable alternative to real-timestability testing, and can accurately predict the shelf life ofbiological products. Such “accelerated stability testing” conditions areknown in the art and are in accordance with the International Conferencefor Harmonization of Technical Requirements for Registration ofPharmaceuticals for Human Use: Stability Testing of New Drug Substancesand Products Q1A, herein incorporated by reference in its entirety.

One method of accelerated stability testing comprises storing samples ofdigestive enzyme composition in a sealed Nialene (nylon, aluminium,polyethylene laminate; GOGLIO SpA, Milan) bag at 40° C./75% relativehumidity for 6 months.

After storage (or periodically during storage) the enzyme activity ofthe samples can be tested using conventional methods for assayingdigestive enzyme activity (e.g., United States Pharmacopoeia,Pancrelipase: Assay for lipase activity; herein incorporated byreference in its entirety).

The compositions or oral dosage forms of the present invention can alsofurther comprise one or more stabilizers which enhance or improve thestability of the compositions or oral dosage forms of the presentinvention. Non-limiting examples of suitable stabilizers includeproline, trehalose, dextran, maltose, sucrose, mannitol, polyols, silicagel, aminoguanidine, pyridoxamine, anhydrous metal salts, such as sodiumhydrogen carbonate magnesium oxide, calcium oxide, aluminium oxide andmixtures thereof. The one or more stabilizers can have a moisturecontent of about 3% or less and/or a water activity of 0.6 or less.

Non-limiting examples of suitable forms of trehalose which can be usedin the compositions or oral dosage forms of the present inventioninclude trehalose dihydrate (TD), amorphous trehalose (AT), anhydroustrehalose (e.g. anhydrous amorphous trehalose (AAT), anhydrouscrystalline trehalose (ACT)). Powdered anhydrous trehalose may containany AAT and/or ACT. As used herein, the term “trehalose” refers to anyphysical form of trehalose, including anhydrous, partially hydrated,fully hydrated and mixtures and solutions thereof. The term “anhydroustrehalose” refers to any physical form of trehalose containing less than2% water. The anhydrous forms of trehalose may contain from 0-2% water.Amorphous trehalose contains about 2-9% water and trehalose dihydratecontains about 9-10% water. Anhydrous trehalose can be prepared asdescribed in PCT/GB97/00367, herein incorporated by reference in itsentirety. In one embodiment, the compositions or oral dosage forms ofthe present invention comprise one or more stabilized digestive enzymesand anhydrous trehalose.

The amount of anhydrous trehalose (AAT or ACT) in the composition of thepresent invention can be in the range of about 5-50%, 5-40%, 5-30%,5-20%, 5-15%, 5-10%, 7-15%, or about 5%, about 7%, about 10%, about 15%,or about 20%.

The anhydrous trehalose can be incorporated into the compositions ororal dosage forms of the present invention as a powder. The particlesize of the anhydrous trehalose powder can be in the range of about2-2000 μm.

Compositions or oral dosage forms of the present invention comprisingone or more stabilized digestive enzymes and anhydrous trehalose conferimproved enzyme stability. It is believed that the anhydrous trehalosestabilizes the compositions or oral dosage forms of the presentinvention by absorbing or sequestering moisture from ambient humidity,or residual moisture from manufacturing or within the formulationitself.

Depending on the intended use and requirement of the compositions, theweight ratio of the stabilized digestive enzyme to the stabilizer rangesfrom about 99:1 to 80:20. The stabilizer can be incorporated into thecompositions or oral dosage forms of the present invention by wet or dryblending at least one stabilized digestive enzyme with at least onestabilizer. In one embodiment, one or more stabilized digestive enzymeis dry blended with one or more stabilizer. In another embodiment, oneor more stabilized digestive enzyme is wet blended with one or morestabilizer.

In addition to the stabilized digestive enzyme and/or stabilizer(s), thecompositions or oral dosage forms of the present invention can furthercomprise one or more pharmaceutically acceptable excipients. The term“excipients” includes other pharmaceutically acceptable ingredientsadded to the active component(s) of a composition (e.g., the stabilizeddigestive enzymes) in order to improve processing, stability,palatability, etc. Non-limiting examples of suitable excipients includepharmaceutically acceptable binders, stabilizers, disintegrants,lubricants, glidants, diluents, and mixtures thereof etc. It will beappreciated by those skilled in the art of pharmaceutical formulationsthat a particular excipient may carry out multiple functions in thecomposition. So, for example a binder may also function as a diluent,etc. The excipients can have a moisture content of about 3% or lessand/or a water activity of about 0.6 or less.

Non-limiting examples of suitable binders include starches, sugars (e.g.lactose), sugar alcohols (e.g. xylitol, sorbitol, maltitol), cellulose(e.g. microcrystalline cellulose), modified celluloses (e.g.,hydroxypropylcellulose, carboxymethylcellulose sodium), alginic acid,polyvinyl pyrrolidone (povidone), and mixtures thereof. Non-limitingexamples of suitable disintegrants include dibasic calcium phosphate,dibasic calcium phosphate dihydrate, tribasic calcium phosphate, alginicacid, hydroxypropylcellulose, carboxymethylcellulose calcium,carboxymethylcellulose sodium, cross-linked carboxymethylcellulosesodium, swellable ion exchange resins, alginates, formaldehyde-casein,cellulose, croscarmellose sodium, crospovidone (e.g., cross-linkedpolyvinyl pyrrolidone), microcrystalline cellulose, sodium carboxymethylstarch, sodium starch glycolate, starches (corn starch, rice starch),and mixtures thereof. Non-limiting examples of suitable lubricantsinclude calcium stearate, magnesium stearate, sodium stearyl fumarate,stearic acid, zinc stearate, talc, waxes, Sterotex®, Stearowet®, andmixtures thereof. Non-limiting examples of suitable glidants includecolloidal silicon dioxide, talc, and mixtures thereof. Non-limitingexamples of suitable diluents include mannitol, sucrose, anhydrousdibasic calcium phosphate, anhydrous dibasic calcium phosphatedihydrate, tribasic calcium phosphate, cellulose, lactose, magnesiumcarbonate, microcrystalline cellulose, and mixtures thereof.Non-limiting examples of suitable stabilizers include trehalose,proline, dextran, maltose, sucrose, mannitol, polyols, silica gel,aminoguanidine, pyridoxamine, and mixtures thereof.

In one embodiment, the disintegrant is crospovidone (e.g., POLYPLASDONEXL, POLYPLASDONE XL-10). In another embodiment, the disintegrant iscroscarmellose sodium (e.g., AC-DI-SOL). In another embodiment, thedisintegrant is sodium starch glycolate (e.g., EXPLOTAB, EXPLOTAB CV).In another embodiment, the compositions or oral dosage forms of thepresent invention can comprise a combination of disintegrants such asmicrocrystalline cellulose and sodium starch glycolate or croscarmellosesodium and crospovidone.

The amount of disintegrant can be in the range of about any of about0.1-30%, 1%-30%, 1%-25%, 1%-20%, 1%-15%, 1%-10%, 1%-5%, 5%-10%, 5%-15%,5%-20%, 5%-25%, or 5%-30%. In one embodiment, the amount of disintegrantis about 2%-4%, or about 2%-3%, or about 2.5%.

Non-limiting examples of suitable diluents include microcrystallinecellulose, starch, calcium phosphate, lactose, sucrose, mannitol,sorbitol, and combinations thereof. In one embodiment, the diluent ismicrocrystalline cellulose (e.g. Avicel). In another embodiment, thediluent is starch. In another embodiment, the diluent is lactose (e.g.,Pharmatol). In another embodiment, the compositions or oral dosage formsof the present invention can comprise a combination of diluents such asmicrocrystalline cellulose, starch and lactose.

The amount of diluent can be in the range of about any of about 0.1-99%,1%-30%, 1%-25%, 1%-20%, 1%-15%, 1%-10%, 1%-5%, 5%-10%, 5%-15%, 5%-20%,5%-25%, or 5%-30%. In one embodiment, the amount of diluent is about2%-5%, 3%-5%, or about 4%.

One or more of the excipients of the compositions or oral dosage formsof the present invention can function as a dessicant to furtherstabilized the composition. Suitable excipients useful as desiccantsinclude any pharmaceutically acceptable excipient that binds watertightly, or reduces the water activity of a composition. For example,the composition of the present invention can include about 1-4% silicagel, or about 2.5% silica gel.

The compositions of the present invention can be prepared in anysuitable oral dosage form. Non-limiting examples of suitable dosageforms include tablets, capsules or sachets. Since certain digestiveenzymes, such as pancreatic lipases may need to be protected againstgastric inactivation prior to release in the duodenum, it is may bedesirable that the stabilized digestive enzyme compositions or oraldosage forms of the present invention be provided as a controlled ordelayed release formulation. Such controlled or delayed releaseformulations can include tablets or particles coated with an entericcoating which serves to protect pH-sensitive digestive enzymes fromgastric inactivation, yet which releases the digestive enzymes in theduodenum. Alternatively, the controlled release formulations can includecapsules filled with the stabilized digestive enzyme compositions ororal dosage forms of the present invention, whereby the capsule protectsthe contents against gastric inactivation, yet releases the digestiveenzymes in the duodenum. However, the stabilized digestive enzymecompositions or oral dosage forms of the present invention are notlimited to digestive enzymes susceptible to gastric inactivation, forexample certain digestive enzymes that are naturally stable in thegastric environment such as gastric lipases, a range or proteases,including those of pancreatic origin and amylases. Certain digestiveenzymes derived or extracted from microorganisms that have an intrinsicstability, or that have been chemically modified by cross-linking.

When the compositions of the present invention are formulated astablets, the stabilized digestive enzyme(s) can be “tabletted” (i.e.,formed into tablets) using methods known in the art, and subsequentlycoated with an enteric coating, again using methods known in the art.

When the compositions of the present invention are formulated ascapsules, the contents of the capsule can be formulated using methodsknown in the art. For example, the stabilized digestive enzymecomposition can be provided in the form of particles or tablets suitedto incorporation in a capsule.

The term “particles” as used herein includes fine powders (havingparticle diameters in the range of about 1 μm) up to pellets having adiameter of about 5 mm.

The stabilized digestive enzyme composition can also be formed intoparticles coated with a coating, wherein the coating comprises anenteric polymer. The term “enteric polymer” means a polymer thatprotects the digestive enzymes from gastric contents, for example apolymer that is stable at acidic pH, but can break down rapidly athigher pH or a polymer whose rate of hydration or erosion is slow enoughto ensure that contact of gastric contents with the digestive enzymes isrelatively minor while it is in the stomach, as opposed to the remainderof the gastro-intestinal tract. Non-limiting examples of entericpolymers include those known in the art, such as modified or unmodifiednatural polymers such as cellulose acetate phthalate,hydroxypropylmethylcellulose phthalate, hydroxypropylmethylcelluloseacetate succinate, and shellac; or synthetic polymers such as acrylicpolymers or copolymers methacrylic acid polymers and copolymers,methylmethacrylate copolymers, and methacrylic acid/methylmethacrylatecopolymers.

The enteric polymer coating can be a synthetic polymer, optionallyincluding an inorganic material, such as an alkalinizing agent. Theresulting coated particles provide delayed release beads comprising acore which comprises the stabilized digestive enzyme(s) and an entericcoating encapsulating the core. The coated stabilized digestive enzymeparticles can then be formulated into tablets or capsules.

The enteric polymer and the at least one inorganic material impartenteric properties to the coating of the present invention. That is,when used as a medication, the coating will act as a barrier protectingthe medication from the acidic environment of the stomach andsubstantially prevent the release of the medication before it reachesthe small intestine (i.e., the release of enzyme in the stomach is lessthan about 10-20% of the total amount of enzyme in the composition).

The inorganic material can include, for example, an alkalinizing agent.Non-limiting examples of alkalinizing agents include silicon dioxide,sodium salts, calcium salts, magnesium salts, aluminum salts, aluminumhydroxides, calcium hydroxides magnesium hydroxides, talc, andcombinations thereof. In one embodiment, the alkalinizing agent is talc.

Depending on the intended use of the composition, the ratio of theenteric polymer and the at least one inorganic material may be in arange of from about 10:1 to about 1:60 by weight. In another embodiment,the ratio of the enteric polymer and the at least one inorganic materialranges from about 8:1 to about 1:50 by weight. In another embodiment,the ratio of the enteric polymer and the at least one inorganic materialranges from about 6:1 to about 1:40 by weight. In another embodiment,the ratio of the enteric polymer and the at least one inorganic materialranges from about 5:1 to about 1:30 by weight. In another embodiment,the ratio of the enteric polymer and the at least one inorganic materialranges from about 4:1 to about 1:25 by weight. In another embodiment,the ratio of the enteric polymer and the at least one inorganic materialranges from about 4:1 to about 1:9 by weight. In another embodiment, theratio of the enteric polymer and the at least one inorganic materialranges from about 10:4 to about 10:7 by weight.

In one embodiment, the compositions or oral dosage forms of the presentinvention comprise stabilized digestive enzyme particles coated with anenteric coating comprising an enteric polymer and an inorganic materialsuch as talc. In a particular embodiment, the inorganic material of theenteric coating comprises about 1-10% by weight of the weight of thetotal weight of the particles. In another embodiment the inorganicmaterial comprises about 3, about 5, about 7, or about 10% by weight ofthe particles. In still other embodiments, the inorganic material is analkalinizing agent and comprises about 20-60% of the dry coating weight.In still other embodiments, the alkalinizing agent is about 25%, about30%, about 35%, about 40%, about 45%, about 50%, or about 55% of the drycoating weight (inclusive of all ranges, subranges, and valuestherebetween). In a particular embodiment, the alkalinizing agent istalc. In still another particular embodiment, the dry coating of theparticles comprises about 35% talc.

In another embodiment of the present invention, the coating furthercomprises a plasticizer. Examples of suitable plasticizers include, butare not limited to triacetin, tributyl citrate, tri-ethyl citrate,acetyl tri-n-butyl citrate, diethyl phthalate, dibutyl sebacate,polyethylene glycol, polypropylene glycol, castor oil, acetylatedmono-glyceride, acetylated di-glyceride, and mixtures thereof.

The dosage forms of the present invention can be capsules containing thecomposition of the present invention (e.g., controlled-release particlesof the stabilized digestive enzyme composition, coated with an entericpolymer and an alkalinizing agent). The capsules themselves can becomprised of any conventional biodegradable material known in the art,for example, gelatin, polysaccharides such as pullulan, or modifiedcellulosic materials such as hydroxypropylmethylcellulose. In order toimprove the stability of the stabilized digestive enzymes, the capsulecan be dried prior to filling, or a capsule comprised of a low moisturecontent material can be selected. In one embodiment of the dosage formof the present invention, the capsule is comprised ofhydroxypropylmethylcellulose. In another embodiment of the dosage formof the present invention, the capsule is comprised ofhydroxypropylmethylcellulose having a water content of about 6% or less,for example about any of 4% or less, 2% or less, or 2-6%, or 4-6%. Inanother embodiment, the capsule is comprised ofhydroxypropylmethylcellulose having a water content of less than about2%.

The dosage forms of the present invention can comprise a singledigestive enzyme, or mixtures of digestive enzymes. If the stabilizeddigestive enzyme composition is formed into particles coated with anenteric coating, the coated particles can each contain a core comprisinga single digestive enzyme or mixtures of digestive enzymes. The dosageform can also comprise coated particles, each of which has nominally thesame composition, or it can comprise mixtures of different kinds ofcoated particles. For example the dosage form can be a capsule filledwith coated particles, wherein each of the coated particles has a corecomprising pancrelipase. Alternatively, the dosage form can be a capsulefilled with coated particles, wherein some of the coated particles havea core comprising pancrelipase, whereas other coated particles havecores comprising a different lipase, or proteases or amylases. Anysuitable combination of coated particles of different compositions canbe used to provide the desired therapeutic effect.

In addition, when the dosage forms of the present invention arecomprised of coated particles of stabilized digestive enzymes, theindividual particles can each have the same coating composition, or caninclude mixtures of particles, some of which have a different coatingcomposition. Any suitable combination of coating compositions can beused to provide the desired type of controlled release or therapeuticeffect.

The core of the coated particles can have any suitable particle size orshape. For example, the coated particles can be in the form of a coatedpowder having a particle size range of about 50-5000 microns, or can bein the form of “minitabs” which have a nominal particle diameter in therange of about 2-5 mm. For other applications, the core of the coatedparticles can be “microtabs” which have nominal particle diameters ofless than about 2 mm, for example about 1-2 mm.

In one embodiment, the compositions or oral dosage forms of the presentinvention can comprise a plurality of coated digestive enzyme particles(e.g., pancrelipase). The digestive enzyme particles can comprise adigestive enzyme, at least one disintegrant, at least one polymericbinder or diluent, and optionally at least one plasticizer, optionallyat least one glidant, and optionally at least one lubricant. In oneembodiment, the digestive enzyme particles can comprise about 60-90% ofdigestive enzyme, about 1-4% of at least one disintegrant, about 2-6% ofat least one polymeric binder or diluent, and optionally about 0.5-1.0%of at least one plasticizer, optionally about 0.2-0.6% of at least oneglidant, and optionally about 0.2-0.6% of at least one lubricant. Forexample, the digestive enzyme particles can comprise about 60-90%pancrelipase, about 1-4% of croscarmellose sodium, about 0.5-1.0% ofhydrogenated castor oil, about 0.2-0.6% of colloidal silicon dioxide,about 2-6% of microcrystalline cellulose, and about 0.2-0.6% ofmagnesium stearate. The coating can comprise at least one entericpolymer, about 4-10% of at least one alkalinizing agent (based on thetotal weight of the particles), and optionally at least one plasticizer.In one embodiment, the coating can comprise about 10-20% of a least oneenteric polymer, about 4-10% of a least one alkalinizing agent, andabout 1-2% of a least one plasticizer (based on the total weight of theparticles). For example, the coating can comprise about 10-20% ofhydroxypropylmethylcellulose phthalate, about 4-10% of talc, and about1-2% of triethyl citrate (based on the total weight of the particles).The plurality of coated digestive enzyme particles can then be formedinto a tablet, or filled into a capsule. In one embodiment, the capsulecomprises hydroxypropylmethylcellulose.

The compositions of the present invention, and dosage forms comprisingthe compositions of the present invention, have improved stabilitycompared to conventional digestive enzyme (e.g., pancrelipase)compositions and dosage forms. Consequently, the dosage forms of thepresent invention do not require “overfilling” (i.e., zero-overfill), asdo conventional digestive enzyme dosage forms, to deliver a clinicallyuseful amount of digestive enzyme to a patient in need thereof.Conventional digestive enzyme compositions and dosage forms requireoverfilling levels of as much as 65% (i.e., 165% of the required dose ofdigestive enzyme) to compensate for the poor enzyme stability. As aresult, there is uncertainty as to the dose delivered by conventionaldigestive enzyme compositions. Thus, conventional “overfilled” dosageforms can deliver higher than the intended dose of digestive enzymesshortly after manufacture, but over time, the enzyme activity can fallbelow the intended dose.

In one embodiment, the dosage forms comprising the compositions of thepresent invention are substantially zero-overfill. The term“substantially zero-overfill” means compositions of the presentinvention in which the amount of additional digestive enzyme activity(i.e., the amount of additional enzyme activity above the intended dose)is less than or equal to about 10%, i.e., about 10%, less than about10%, less than or equal to about 9%, less than or equal to about 8%,less than or equal to about 7%, less than or equal to about 6%, lessthan or equal to about 5%, less than or equal to about 4%, less than orequal to about 3%, less than or equal to about 2%, less than or equal toabout 1%, or about 0%. So, for example, if the intended dose is about4500 IU lipase, the substantially zero-overfill dosage forms of thepresent invention may contain less than or equal to about 4950 IU lipase(i.e., less than or equal to 110% of 4500 IU lipase). In anotherembodiment, the zero-overfill dosage form contains 4500 IU lipase.

The compositions or dosage forms (e.g., tablets or capsules) of thepresent invention can be stored in any suitable package. For example,the package can be a glass or plastic jar with a threaded or press-fitclosure. Alternatively, the compositions or dosage forms of the presentinvention can be packaged as a unit dosage form in “blister packs”.Applicants have found that improved stability of the digestive enzymecompositions or dosage forms can be provided by providing amoisture-proof seal, and/or a moisture-proof package. Non-limitingexamples of suitable moisture-proof packages include glass jars, plasticjars incorporating moisture barrier resins or coatings, aluminizedplastic (e.g., Mylar) packaging, etc. The term “moisture-proof” refersto a package which has a permeability to water of less than about 0.5 mgwater per cm³ of container volume per year.

Containers (e.g., bottles) can be closed with any suitable closure,especially closures which minimize the ingress of moisture duringstorage. For example, the compositions or dosage forms of the presentinvention can be closed with a closure such as Saf-Cap III-A (VanBlarcom Closures, Inc.), containing HS 035 Heat Seal/20F (SANCAP LinerTechnology, Inc.) printed as a sealing liner.

In order to ensure package integrity and minimize moisture ingressduring storage, sealed packages containing the compositions or dosageforms of the present invention can be leak-tested after dispensing thecomposition or dosage form of the present invention and sealing thepackage. For example, the sealed packages can be tested by applying acontrolled vacuum to the closure, and detecting the decrease in vacuumover time. Suitable leak-testing equipment includes those manufacturedby Bonfiglioli (e.g., model LF-01-PKV or model PKV 516).

Packages containing the compositions or dosage forms of the presentinvention can also contain a desiccant (i.e., a substance which absorbs,reacts with, or adsorbs water) capable of reducing the humidity insidethe package, for example a desiccant capsule, capable of “scavenging”moisture from the atmosphere sealed inside the package. Non-limitingexamples of suitable dessicants which can be placed inside such packagesinclude zeolites (e.g., molecular sieves such as 4 Å molecular sieves),clay (e.g., montmorillonite clay), silica gel, activated carbon, orcombinations thereof. In one embodiment, the desiccant comprisesmolecular sieves.

In addition, it is common practice when packaging oral pharmaceuticalunit doses to add a “plug” of a cellulosic material, such as cotton,into the top of the container to fill the empty space at the top of thecontainer, thereby minimizing movement of the contents. Cellulosicmaterials are somewhat hygroscopic, and can act as a “reservoir” ofmoisture inside the package. Accordingly, in one embodiment of thepackages of the present invention, no cellulosic or cotton “plug” ispresent in the package. In another embodiment of the packages of thepresent invention, the packages lack a cellulosic or cotton plug, andcontain a desiccant.

The compositions of the present invention can be prepared usingconventional techniques, but modified as indicated herein to providemoisture contents of about 3% or less, water activities of about 0.6 orless, or provide stabilized digestive enzyme compositions which exhibita loss of activity of no more than about 15% after three monthsaccelerated stability testing. For example, particles of digestiveenzymes (e.g., pancrelipase) can be coated in a fluidized bed coatingapparatus equipped with a dehumidifier. In one embodiment, the coatingapparatus is operated in an atmosphere having a water content of about 4g/m³ or less, about 3.5 g/m³ or less, about 3 g/m³ or less, about 2.5g/m³ or less, about 2.0 g/m³ or less, about 1.5 g/m³ or less, about 1.0g/m³ or less, or about 0.5 g/m³ or less, including all ranges andsubranges therebetween. The atmosphere in which the coating is carriedout can comprise dehumidified air, dehumidified nitrogen, or anotherdehumidified inert gas.

The coating can be applied as a solution of the enteric polymer (andoptionally a suspended inorganic material) in an organic solvent such asan alcohol (e.g. ethanol), a ketone (e.g. acetone), methylene chloride,or mixtures thereof (e.g. mixtures of acetone ethanol).

The compositions of the present invention provide improved absorption offats, proteins, and carbohydrates in patients suffering from conditionsor disorders associated with a digestive enzyme deficiency. In oneembodiment, compositions of the invention, in particular pancrelipase orpancreatin compositions, may be used to treat exocrine pancreaticinsufficiency (EPI) associated with various diseases. Such diseasesinclude, but are not limited to cystic fibrosis (CF). In someembodiments, such compositions may substantially alleviate malabsorption(e.g. of fats) associated with EPI in cystic fibrosis patients and otherpatients, including pediatric patients. In some embodiments, suchcompositions may increase the coefficient of fat absorption (CFA) to atleast about 85% or more in cystic fibrosis patients. Such results may beachieved when co-administered with other agents or compositions, or maybe achieved without co-administration with other agents. In oneembodiment, such CFA results are achieved without co-administration ofproton pump inhibitors such as Prilosec®, Nexium®, and the like.

For patients identified as having low GI pH levels (e.g., GI pHlevels<about 4), improved results may be obtained by administering thecompositions or dosage forms of the present invention together withproton pump inhibitors, antacids, and other drugs which increase the pHof the GI tract. For example, the compositions or dosage forms of thepresent invention can be administered separately from the proton pumpinhibitors, antacid, or other drugs (either prior to, concurrently with,or after administration of the proton pump inhibitor, antacid, etc.).Alternatively, the proton pump inhibitor, antacid, or other drug can becombined with the pancreatin composition of the present invention as asingle dosage form.

In yet another embodiment, the present invention provides a method oftreating or preventing a disorder associated with a digestive enzymedeficiency comprising administering a composition of the presentinvention to a mammal in need thereof. In one embodiment, the mammal isa human.

In yet another embodiment, the present invention provides a method oftreating or preventing a disorder associated with a digestive enzymedeficiency comprising administering a composition or dosage form of thepresent invention to a mammal in need thereof, wherein the compositionor dosage form of the present invention comprises, in addition to atleast one digestive enzyme, a proton pump inhibitor, antacid, or othermedicament which increases GI pH. In still another embodiment, thepresent invention provides a method of treating or preventing a disorderassociated with a digestive enzyme deficiency, comprising administeringa composition or dosage form of the present invention, in combinationwith a dosage form comprising a proton pump inhibitor, antacid, or othermedicament which increases GI pH.

Disorders which can be treated with the composition or dosage form ofthe present invention include conditions in which the patient has no orlow levels of digestive enzymes or in which patients require digestiveenzyme supplementation. For example, such conditions can include cysticfibrosis, chronic pancreatitis, other pancreatic diseases (e.g.,hereditary, post-traumatic and allograft pancreatitis, hemochromatosis,Shwachman syndrome, lipomatosis, or hyperparathyroidism), side-effectsof cancer or cancer treatment, side-effects of surgery (e.g.,gastrointestinal bypass surgery, Whipple procedure, totalpancreatectomy, etc.) or other conditions in which pancreatic enzymescannot reach the intestine, poor mixing (e.g., Billroth II gastrectomy,other types of gastric by pass surgery, gastrinoma, etc.) side effectsof drug treatments such as treatment with metformin or those drugs usedto treat the symptoms of HIV and autoimmune diseases such as diabetes inwhich the pancreas may be compromised, obstruction (e.g., pancreatic andbiliary duct lithiasis, pancreatic and duodenal neoplasms, ductalstenosis), malabsorption associated with celiac disease, food allergiesand aging.

The amount of the composition or dosage form of the present inventionadministered daily to mammals (e.g., humans) depends upon the intendedresult. The skilled physician will be capable of prescribing therequired dose based on his diagnosis of the condition to be treated.

For example, for the treatment of digestive enzyme insufficiency inhumans (e.g., related to cystic fibrosis) the starting dose should be500 to 1000 lipase units/kg/meal, with the total dose not exceeding 2500lipase units/kg/meal or 4000 lipase units/g fat/meal in accordance withthe recommendations of the US FDA. Typically, a patient should receiveat least 4 dosage forms per day, preferably administered with food.

EXAMPLES Example 1

Pancrelipase MT (minitablets) is a blend of pancrelipase raw material(e.g., obtained from Nordmark) and excipients (e.g., croscarmellosesodium, hydrogenated castor oil, colloidal silicon dioxide,microcrystalline cellulose, and magnesium stearate; Table 34) tablettedusing round 2 mm diameter beveled punches. The physical characteristicsof the Pancrelipase MT before coating are shown below in Table 1.

TABLE 1 Diameter 2.0 mm Weight (of 10 MT) 0.074-0.086 g Thickness (meanvalue of 10 MT) 2.2 ± 0.2 mm Hardness 0.5-2.0 Kp Friability* (20 g ofMT-30 min at 25 rpm) 0.0-2.5% *USP method

Pancrelipase MT was coated with a coating formulation (Table 2) using afluidized bed Glatt-GPCG1 apparatus equipped with a Munters ML 1350dehumidifier in the process airflow. The coating process was carried outwith process air at three different moisture contents (Table 3). Foreach batch, the coating weight was approximately 15% of the total weightof the coated particles. The composition of the coated particles foreach set a process conditions is approximately the same (Table 4), andappeared uniform, smooth and homogeneous after microscopic examination.

TABLE 2 Material % (w/w) Hypromellose Phthalate (HP55) 10.19 Triethylcitrate (TEC) 1.02 Talc 1.02 Ethanol 96% 79.78 Acetone 7.99 100.00

TABLE 3 Process Air Moisture Content Lot (g/m³) P9A165 8.8 P9A167 0.4P9A170 3.6

TABLE 4 Coating Composition % Material (w/w) Pancrelipase MT 85.00Hypromellose Phthalate (HP55) 12.50 Triethyl citrate (TEC) 1.25 Talc1.25 100.00

The three sets of samples (i.e., P9A165, P9A167, and P9A170) showedresidual moisture contents corresponding to the moisture content of theprocessing air flow (Table 4).

TABLE 5 Loss on Drying Lot (%) P9A165 2.8 P9A167 1.1 P9A170 1.7

The influence of residual moisture on the loss of activity over time wasevaluated under accelerated stability conditions as follows:

Hard gelatin capsules (dosage 20,000 IU Lipase) were filled with thethree lots of coated Pancrelipase MT minitablets described above andstored at 40° C. at 75% relative humidity in sealed Nialene bags.

Lipase activity was evaluated after 15 days and 4 months of storage. Theresults are shown in Table 6.

TABLE 6 Time zero 15 days 4 months Batch LoD Lipase (IU/mg) P9A165(2.8%) 62.5 46 (−26% activity) 33.6 (−46% activity) P9A167 (1.1%) 64.553 (−18% activity) 46.2 (−28% activity) P9A170 (1.7%) 63.8 53 (−17%activity) 44.8 (−30% activity)

The results of Table 6 show that improved stability is provided bycompositions having a moisture content of less than about 2%.Alternatively, improved stability is provided by coating under anatmosphere with a moisture content of less than 3.6 g/m³ to 0.4 g/m³.

Example 2

Pancrelipase MT particles were coated with two coating compositionscontaining different amounts of talc (Table 7).

TABLE 7 Composition % (w/w) Material Low talc content High talc contentHypromellose Phthalate (HP55) 10.190 5.825 Triethyl citrate (TEC) 1.0200.580 Talc 1.020 5.825 Ethanol 96% 79.780 79.780 Acetone 7.990 7.990100.000 100.000 HP:TEC:Talc ratio 10:1:1 10:1:10 Total solid content12.23% 12.23%

Coating trials were carried out using a fluidized bed Glatt-GPCG1apparatus equipped with a Munters ML 1350 dehumidifier in order toassure process air flow at a low moisture content (i.e., lower than 1g/m³). Coating weights were approximately 15%. The theoreticalcomposition of the two batches is reported in Table 8. Microscopicexamination indicated that the coatings on all samples were smooth andhomogeneous. Residual moisture contents were measured by loss on drying(Table 9).

TABLE 8 Batch P9A230 P9A240 Low talc content High talc content MaterialComposition % (w/w) Pancrelipase MT 85.000 85.000 Hypromellose Phthalate(HP55) 12.500 7.143 Triethyl citrate (TEC) 1.250 0.714 Talc 1.250 7.143100.000 100.000

TABLE 9 Loss on Drying Lot (%) P9A230 0.9 P9A240 0.9

The effects of the different coating compositions on the loss ofactivity over time were evaluated under accelerated stability conditionsas follows:

Hard gelatin capsules (dosage 20,000 IU Lipase) were filled with the twolots of coated Pancrelipase MT described above, and stored at 40° C. and75% relative humidity in sealed Nialene bags.

Lipase activity was checked after 1, 2 and 3 months of storage as shownin Table 10.

TABLE 10 Time zero 1 month 2 months 3 months Batch Lipase (IU/mg) P9A23064.5 57.6 49.6  52.3 Low talc (−11% activity) (−23% activity) (−19%activity) content P9A240 65.3 58.2 60.62 59.6 High talc (−11% activity) (−7% activity)  (−9% activity) content

The results showed that the loss of activity after three months ofstorage under accelerated stability conditions is significantly lowerfor samples coated with a high talc content coating (Lot P9A240).Accordingly, increasing the talc concentration from approximately 1% toapproximately 7% results in significant improvements in enzymestability.

Example 3

The effects of coating composition solvent was evaluated by preparing“high talc” and “low talc” coating compositions similar to thosedescribed in table 6, except that the ethanol (96% ethanol, 4%water)/acetone solvent was replaced with 100% acetone (Table 11).

TABLE 11 Composition % (w/w) Material Low talc content High talc contentHypromellose Phthalate (HP55) 10.190 5.825 Triethyl citrate (TEC) 1.0200.580 Talc 1.020 5.825 Acetone 87.770 87.770 100.000 100.000 HP:TEC:Talcratio 10:1:1 10:1:10 Total solid content 12.23% 12.23%

The coating trials were carried out using a fluidized bed Glatt-GPCG1apparatus equipped with a Munters ML 1350 dehumidifier in order toassure process air flow at a low moisture content (lower than 1 g/m³).Coating weights were approximately 15%. The theoretical composition ofthe two batches is reported in Table 12.

TABLE 12 Batch P9A318 P9A352 Low talc content High talc content MaterialComposition % (w/w) Pancrelipase MT 85.000 85.000 Hypromellose Phthalate(HP55) 12.500 7.143 Triethyl citrate (TEC) 1.250 0.714 Talc 1.250 7.143100.000 100.000

Lot P9A318 complied with commercial product specifications, but LotP9A352 did not pass a gastro-resistance test. Microscopic examinationshowed that the film coating of Lot P9A352 was not as smooth andhomogeneous as other coated samples, probably because of the higherevaporation rate of acetone compared with the ethanol/acetone mixtureused in previous samples, and the high talc concentration in thecoating.

Lot P9A318 was then evaluated under accelerated stability conditions asfollows:

Hard gelatin capsules (dosage 20,000 IU Lipase) were prepared and storedat 40° C. and 75% relative humidity in sealed Nialene bags. Lipaseactivity was measured after 1, 2 and 3 months of storage as shown inTable 13.

TABLE 13 Time zero 1 month 2 months 3 months Batch Lipase (IU/mg) P9A31863.6 59.5 60.4 55.4 Low talc (−6% activity) (−5% activity) (−13%activity) content

The stability of Lot P9A318 is significantly improved compared to thestability of Lot P9A230, which was prepared with a similar coating undersimilar coating conditions (Table 14). It therefore appears thatreplacement of 96% ethanol with acetone in the coating formulationprovides a significantly lower loss of enzyme activity over time.

TABLE 14 Accelerated stability at 40° C. + 75% R.H. 1 month 2 months 3months Lot HP:TEC:Talc Talc content Solvent Lipase (loss of activity)P9A230 10:1:1 Low Ethanol\Acetone −11% −23% −19% P9A240 10:1:10 HighEthanol\Acetone −11% −7% −9% P9A318 10:1:1 Low Acetone −6% −5% −13%

Example 4

CPS gelatin and HPMC (hydroxypropylmethylcellulose) capsules were filledwith identical coated lipase compositions. The water content of gelatincapsules is approximately 14%, and the water content of HPMC capsules isapproximately 4%. In addition one set of HPMC capsules was dried to amoisture level of less than 2%. All samples were subjected toaccelerated stability conditions (40° C. and 75% relative humidity;samples heat-sealed in Nialene bags, and lipase activity was testedafter 15, and 90 days. The results are shown below in Tables 15-17.

1) HPMC CPS vs Gelatin CPS

TABLE 15 LOT P200450287 LOSS OF LIPASE ACTIVITY % CPS HPMC (not TIME CPSGELATIN dried) 15 days −12% −3% 30 days −21% −13%

TABLE 16 LOT P200450614 LOSS OF LIPASE % HPMC CPS TIME GELATINE CPS(dried) 30 days −11 −1

TABLE 17 LOT P200450653 LOSS OF LIPASE % HPMC CPS (not TIME GELATINE CPSdried) 30 days −14 −8 90 days −32 −18

As shown in Tables 15-17, lipase compositions in HPMC capsules showsignificantly higher lipase activity after storage for 15, 30, and 90days under accelerated stability conditions and dried HPMC capsulesoffer better stability than those which contain equilibrium moisturelevels.

Example 5

Gelatin and hydroxypropylmethylcellulose capsules were filled withcoated lipase compositions Minitablet form. The coating for thecompositions of the gelatin capsules (P200050) contained approximately10% talc, whereas the coating for the compositions of thehydroxypropylmethylcellulose capsules (P200550) contained approximately33% talc. The coating compositions were otherwise identical. Thefollowing Table 18 compares the levels of degradation observed afterstorage under accelerated stability conditions with the moisture contentof the compositions. As shown in Table 18, higher levels of lipaseactivity correlate with lower levels of moisture in the composition. Inaddition, compositions filled in HPMC capsules are more stable thancompositions filled in to gelatin capsules.

TABLE 18 % activity LOD % months 40° C./75% RH months 40° C./75% RH 0 13 6 0 1 3 6 HPMC Batch P200550 503 100 100 105 101 1.6 1.7 1.6 1.5P200550 865 100 96 101 102 1.7 2.1 1.6 1.8 P200550 500 100 102 101 980.8 1.9 1.7 2 P200550 861 100 97 103 99 1.5 1.7 2.0 1.4 P200550 502 100100 99 98 0.4 1.4 2.3 2.0 P200550 859 100 103 103 97 1.1 0.7 1.9 1.3Average 100 100 102 99 1.2 1.6 1.9 1.7 Gelatin Batch P200050 981 100 9092 81 2.9 3.0 3.0 2.8 P200050 975 100 89 79 66 2.7 3.2 3.1 2.8 P200050977 100 96 93 87 3.2 3.4 3.2 2.9 Average 100 92.5 86 77 3.0 3.3 3.2 2.9

Example 6

The effects of storing capsules containing lipase compositions inpackages containing a desiccant were evaluated by measuring lipaseactivity in the samples after 30 and 90 days of storage underaccelerated stability conditions (40° C. and 75% relative humidity;samples heat-sealed in Nialene bags). As shown in Tables 19 and 20,lipase activity is significantly higher in packages containing adesiccant and in capsules that are dried below their equilibriummoisture content.

2) Desiccants

Desiccant 1: silica gel in Tyvek® bagsDesiccant 2: molecular sieves in Tyvek® bags

TABLE 19 LOSS OF LIPASE % P200450614 in P200450614 in P200450614 in HPMCcps (dried) HPMC cps (dried) HPMC cps (dried) TIME no desiccantdesiccant 1 desiccant 2 30 days −1 +4 +1 90 days −10 +2 0

TABLE 20 LOSS OF LIPASE % P200450653 in P200450653 in HPMC cps noP200450653 in HPMC cps TIME desiccant HPMC cps desiccant 1 desiccant 230 days −8 −8 −5 90 days −18 −14 −10

Example 7

Pancrelipase MT particles were coated with two coating compositionshaving a level of talc intermediate between the “low” and “high” levelsemployed above (HP55:TEC:Talc=10:1:5), using either acetone or a mixtureof ethanol/acetone as the coating solvent. The theoretical compositionof the two coating suspensions shown in Table 21, below.

TABLE 21 Composition % (w/w) Material Intermediate talc contentHypromellose Phthalate (HP55) 7.644 7.644 Triethyl citrate (TEC) 0.7640.764 Talc 3.822 3.822 Ethanol 79.780 Acetone 7.990 87.770 100.000100.000 HP:TEC:Talc ratio 10:1:5 10:1:5 Total solid content 12.23%12.23%

The coating trials were carried out using a fluidized bed Glatt-GPCG1apparatus equipped with a Munters ML 1350 dehumidifier in order toassure process air flow at a low moisture content (lower than 1 g/m³).

The batches were prepared by coating the Pancrelipase MT at a coatingweight of approximately 15%. Three batches were prepared with anethanol/acetone coating solvent and three batches were prepared with anacetone coating solvent. The theoretical composition, which was the samefor all six batches, is shown below in Table 22.

TABLE 22 Batch P9A483-P9A485- P9A486 P9A405-P9A476- Ethanol/AcetoneP9A477 as solvent Acetone as solvent Material Composition % (w/w)Pancrelipase MT 85.00 85.00 Hypromellose Phthalate (HP55) 9.37 9.37Triethyl citrate (TEC) 0.94 0.94 Talc 4.69 4.69 100.00 100.00

Microscopic examination of the coating for all six samples appearedsmooth and homogeneous. The coated Pancrelipase MT particles were thenfilled into HPMC capsules and packaged in glass bottles containingdesiccants (molecular sieves). The bottles were then sealed, storedunder accelerated stability conditions and lipase activity was evaluatedat various time periods as indicated below in Table 23.

The packaging conditions for each sample was as follows. Twelve HPMCcapsules (dosage 20,000 IU Lipase) and 1 g of molecular sieves (Minipaxsorbent-Multisorb) as desiccant were put in a 30 mL capacity glassbottle. The bottles were closed with Saf-Cap III-A, containing HS 035Heat Seal/20F printed as a sealing liner and stored at 40° C./75% RH.

TABLE 23 Accelerated stability at 40° C. + 75% R.H. 0 30 120 180 lotSolvent days 20 days days 40 days 60 days 90 days days days P9A483Ethanol\Acetone Lipase U 69.0 67.0 72.4 62.6 64.7 nd nd nd USP/mg % LOD1.0 0.5 0.2 0.2 0.6 nd nd nd Lipase −3%   5% −9% −6% nd nd nd (loss ofactivity) P9A485 Ethanol\Acetone Lipase U 70.0 73.2 65.7 69.8 66.9 nd ndnd USP/mg % LOD 1.1 0.6 0.3 0.6 0.6 nd nd nd Lipase   5% −6%   0% −4% ndnd nd (loss of activity) P9A486 Ethanol\Acetone Lipase U 63.0 61.4 59.762 61.5 nd nd nd USP/mg % LOD 1.6 0.2%  0.6%  0.5%  0.4%  nd nd ndLipase −3% −5% −2% −2% nd nd nd (loss of activity) P9A405 Acetone LipaseU 64.0 63.2 62.9 65.1 65.5 64.7 66.7 63.1 USP/mg % LOD 1.3 0.3 0.3 0.30.4 0.04 0.6 0.2 Lipase −1% −2%   2%   2% 1% 4% −1% (loss of activity)P9A476 Acetone Lipase U 64.9 65.3 62.1 62.4 62.6 58.7 67.0 61.4 USP/mg %LOD 1.2 0.4 1.0 1.3 0.5 1.0 1.0 0.6 Lipase   1% −4% −4% −4% −10% 3% −5%(loss of activity) P9A477 Acetone Lipase U 68.7 71.7 68.0 67.2 69.7 64.473.4 66.2 USP/mg % LOD 1.1 0.2 0.3 1.0 0.0 0.6 0.7 0.4 Lipase   4% −1%−2%   1% −6% 7% −4% (loss of activity)

As shown in Table 23, the three samples prepared with theethanol/acetone coating solvent showed similar losses in lipaseactivity. After 2 months of storage, two of the samples prepared withthe acetone coating solvent did not exhibit any loss of activity, andthe third showed a 4% of reduction of activity. This suggests thatsamples prepared with the acetone coating solvent are more stable thansamples prepared with the ethanol/acetone coating solvent.

Example 8 Microtablets

To provide further choices for dosage formulations were made in whichthe dimensions of the tablets was significantly reduced. Thepancrelipase blend was tabletted with round 1.5 mm diameter, 1.2 mmradius of curvature punches.

The compression parameters were set to obtain microtablets (“μT”) withfriability lower than 2.5% (USP method). The characteristics of Lot9A402 are shown in Table 24.

TABLE 24 Lot P9A402 Values Diameter 1.5 mm Weight (of 20 μT) 0.071 g(0.070-0.073) Thickness (as mean value of 20μT) 1.73 mm (1.70-1.77)Hardness (as mean value of 20μT) 4 Newton (3-5) Friability (20 g ofμT-30 min at 25 rpm) 1.80%

Lot P9A402 was coated in a fluid bed Glatt-GPCG1 apparatus equipped witha Munters ML 1350 dehumidifier in order to assure process air flow atlow moisture content (lower than 1 g/m³) with a suspension having thecomposition shown in Table 2. A coating weight of 22% was obtained.Microscopic examination of the film coatings indicated that all of thesamples appeared smooth and homogeneous.

The theoretical composition of the batch Lot P9A422 is shown in Table25.

TABLE 25 Standard coat Lot P9A422 Composition % (w/w) Pancrelipase MT78.00 Hypromellose Phthalate (HP55) 18.34 Triethyl citrate (TEC) 1.83Talc 1.83 100.000

Two other batches of microtablets were prepared as described above, andtheir properties are shown below in Table 26.

TABLE 26 Characteristics Lot P9A457 Lot P9A459 Diameter 1.5 mm 1.5 mmWeight (of 20 μT) 0.072 g (0.070-0.073) 0.071 g (0.070-0.074) Thickness(as mean value 1.73 mm (1.67-1.83) 1.74 mm (1.69-1.82) of 20μT) Hardness5 Newton (3-6) 5 Newton (4-6) (as mean value of 20μT) Friability 1.99%2.02% (20 g of μT-30 min at 25 rpm)

Pancrelipase microtablets were coated with one of two suspensions havinglevels of talc intermediate between the “high” and “low” levelsdescribed above (HP55:TEC:Talc=10:1:5), using either acetone or amixture of ethanol in acetone as a coating solvent (Table 27).

The six trials were carried out using a fluidized bed Glatt-GPCG1apparatus equipped with a Munters ML 1350 dehumidifier in order toassure process air flow at low moisture content (lower than 1 g/m³).Coating weights were approximately 22%, and microscopic examinationindicated that the coatings were smooth and homogeneous.

TABLE 27 Coated μT Solvent Uncoated μT Lot. P9A460 Acetone Lot. P9A402Lot. P9A458 Acetone Lot. P9A457 Lot. P9A463 Acetone Lot. P9A459 Lot.P9A473 Ethanol/Acetone Lot. P9A402 Lot. P9A466 Ethanol/Acetone Lot.P9A457 Lot. P9A468 Ethanol/Acetone Lot. P9A459

The theoretical compositions of the batches are summarized in Table 28.

TABLE 28 Batch P9A466-P9A468- P9A473 Ethanol/AcetoneP9A458-P9A460-P9A463 as solvent Acetone as solvent Material Composition% (w/w) Pancrelipase MT 78.00 78.00 Hypromellose Phthalate 13.75 13.75(HP55) Triethyl citrate (TEC) 1.37 1.37 Talc 6.88 6.88 100.00 100.00

HPMC cps capsules were filled with the coated microtablets describedabove, and packed in glass bottles containing desiccants (molecularsieves). The bottles were then closed with Saf-Cap III-A, containing HS035 Heat Seal/20F printed as a sealing liner and stored underaccelerated stability conditions (40° C. and 75% relative humidity).Twelve HPMC capsules (dosage 5,000 IU Lipase) and 1 g of molecularsieves (Minipax sorbent-Multisorb) as desiccant were placed in a 30 mLcapacity glass bottle. Lipase activity was measured at 20, 30, 40, and60 days of storage as shown in Tables 29 and 30.

TABLE 29 Accelerated stability at 40° C. + 75% R.H. 0 40 60 90 120 180lot Solvent days 20 days 30 days days days days days days P9A466Ethanol\Acetone Lipase U 64.7 67.0 64.6 63.6 62.3 nd nd nd USP/mg % LOD1.7 2.2 0.4 0.0 0.0 nd nd nd Lipase 4% 0% −2% −4% nd nd nd (loss ofactivity) P9A468 Ethanol\Acetone Lipase U 61.2 59.6 57.7 58.6 58.9 nd ndnd USP/mg % LOD 1.7 0.5 0.4 0.0 0.0 nd nd nd Lipase 4% 0% −2% −4% nd ndnd (loss of activity) P9A473 Ethanol\Acetone Lipase U 59.8 58.9 57.759.4 58.4 nd nd nd USP/mg % LOD 1.8 0.7 0.9 0.0 0.0 nd nd nd Lipase −2%−4% −1% −2% nd nd nd (loss of activity) P9A458 Acetone Lipase U 62.465.4 64.3 62.9 65.0 62.3 65.5 62.6 USP/mg % LOD 3.0 0.1 0.5 0.0 0.0 0.61.3 0.3 Lipase 5% 3% 1% 4% 0% 5% 0% (loss of activity) P9A460 AcetoneLipase U 56.9 58.2 59.2 58.3 60.0 57.6 62.2 56.8 USP/mg % LOD 1.7 0.070.3 0.0 0.0 0.0 0.6 0.2 Lipase 2% 4% 2% 5% 1% 9% 0% (loss of activity)P9A463 Acetone Lipase U 62.7 63.8 62.2 61.5 59.8 54.5 62.6 58.6 USP/mg %LOD 1.6 2.3 0.5 0.0 0.0 0.4 0.6 0.5 Lipase 2% −1% −2% −5% −13% 0% −7%(loss of activity)

TABLE 30 Accelerated stability at 40° C. + 75% R.H. 20 days 30 days 40days 60 days Lot Solvent Lipase (loss of activity) P9A466Ethanol\Acetone +4% 0% −2% −4% P9A468 Ethanol\Acetone −3 −4% −4 −4%P9A473 Ethanol\Acetone −2% −4% −1% −2% P9A458 Acetone +5% +3% +1% +4%P9A460 Acetone +2 +4% +2% +5% P9A463 Acetone +2% −1% −2% −5%

All three samples prepared using an ethanol/acetone coating solventshowed similar behavior and after two months of storage, the loss oflipase activity was 2%-4%. After two months of storage the loss ofactivity of two of the samples prepared using an acetone coating solventshowed no evidence of loss of lipase activity, while the third sampleshowed a 5% decrease in lipase activity. Thus, compositions preparedwith acetone as the coating solvent were more stable than samplesprepared with an ethanol/acetone coating solvent, possibly linked to thewater content of the ethanol used.

The microtablets prepared above were slightly oblong (see Tables 24 and26); the ratio between the microtablet thickness and diameter wasbetween 1.22:1 and 1.15:1.

To further reduce the dimensions of the microtablets, new samples wereprepared with ratios of thickness to diameter ratio nearer to 1:1 (LotQ9A006), are shown below in Table 31.

TABLE 31 Characteristics Lot Q9A006 Diameter 1.5 mm Weight (of 20 μT)0.060 g (0.058-0.062) Thickness (as mean value of 20μT) 1.50 mm(1.45-1.58) Hardness (as mean value of 20μT) 5 Newton (4-6) Friability(20 g of μT-30 min at 25 rpm) 1.63%

Lot Q9A006 was coated with the compositions shown in Table 32 at acoating weight of 22%. The coating trials were carried out using a fluideyes bed Glatt-GPCG1 apparatus equipped with a Munters ML 1350dehumidifier in order to assure processing air flow at low moisturecontent (lower than 1 g/m³).

The theoretical composition of the coated microtablet Lot Q9A019 was thesame as that shown in Table 28. Microscopic examination indicated thatthe coatings were smooth and homogeneous.

TABLE 32 Composition % (w/w) Material Intermediate talc contentHypromellose Phthalate (HP55) 7.644 Triethyl citrate (TEC) 0.764 Talc3.822 Acetone 87.770 100.000 HP:TEC:Talc ratio 10:1:5 Total solidcontent 12.23%

The above examples show that digestive enzyme compositions with improvedstability can be prepared by maintaining low moisture contents and wateractivities in the components of the composition, for example byreplacing aqueous ethanol/acetone coating solvents with acetone, coatingminitablets and microtablets in dehumidified process air flows (e.g.,having moisture contents between 0.4 g/m³ and 3.6 g/m³). In addition,increased levels of inorganic materials in the coating (e.g.,HP55:TEC:Talc ratios ranging from 10:1:1 to 10:1:5), selection of lesshygroscopic capsule materials (e.g., HPMC or dried HPMC), and improvedpackaging techniques (e.g., storage in well-sealed glass bottlescontaining dessicants) provide digestive enzyme compositions and dosageforms with improved stability.

Example 9

The following Table 33 shows accelerated stability testing (in bottles;40° C. and 75% Relative Humidity) of Eudragit coated pancrelipaseMinitabs.

TABLE 33 Batch 1 2 3 4 5 Time (months) 0 1 0 1 0 1 0 1 0 1 Lipase IU23030 15510 24180 15810 23550 16014 23000 16100 23613 17594 % (Vs time0) 100 67 100 65 100 68 100 70 100 74 L.o.D. % 4.0 4.9 3.9 4.6 4.2 4.23.9 4.3 3.3 3.7 (max. 5.0)

The results indicate that conventional enteric coatings such as Eudragitdo not provide stabilized pancrelipase compositions.

Example 10

Examples of dosage forms comprising ER coated beads of varying dosageper capsule, coated as described in previous examples, are shown inTable 34, below:

TABLE 34 Content (mg/capsule) for each Dosage Strength Composition 1Composition 2 Composition 3 Composition 4 Component (μT) (MT) (MT) (MT)μT or MT Pancrelipase 55.7 108.9 163.4 217.8 (5,000 USP (10,000 USP(15,000 USP (20,000 USP units) units) units) units) CroscarmelloseSodium 1.9 3.6 5.5 7.3 Hydrogenated Castor Oil 0.6 1.2 1.8 2.4 ColloidalSilicon Dioxide 0.3 0.6 0.9 1.2 Cellulose Microcrystalline 3.1 6.1 9.112.1 Magnesium Stearate 0.3 0.6 0.9 1.2 Coating Hypromellose Phthalate12.2 18.9 28.4 37.8 Talc 6.1 9.5 14.2 18.9 Triethyl Citrate 1.2 1.92 2.83.8 Acetone^(b) Trace Trace Trace Trace Capsule Carrageenan 0.1 0.2 0.30.3 Potassium Chloride 0.2 0.3 0.4 0.4 Titanium Dioxide 2.3 3.5 5.1 5.2Hypromellose 33.5 52.9 79.4 79.2 Carnauba Wax Trace Trace Trace TraceWater 0.38 0.60 0.9 0.90 Yellow Iron Oxide — 0.1 — 0.2 Red Iron Oxide —— 0.3 — FDC Blue 2 — — — 0.1

Example 11

The following Table 35 shows the water content of various sizedcontainers containing capsules comprising the compositions of thepresent invention. The water content includes total water from thecapsules, and water permeating into the container over a two-yearstorage time. The “equivalent molecular sieves weight” is the minimumamount of molecular sieves required to absorb the water present in thecontainers.

TABLE 35 Bottle Cps Cps. Total water Water from Equivalent sizes weightmoisture from cps Permeation. Molec. Sieves (cc) Cps n^(o) (mg) (%)(mg/bottle) (mg/2y/bottle) weight (g) 30 12 95 3% 34 111 0.96 200 100 953% 285 401 4.58 750 500 95 3% 1425 474 12.66 30 20 95 3% 57 111 1.12

Example 12

A Phase III randomized, double-blind, placebo-controlled, cross overstudy was carried out to compare the effects of treatment with thepancrelipase compositions of Table 34 to that of a placebo in 34 CFpatients with EPI aged seven years and older. The study was conducted in14 CF centers throughout the US. The primary endpoint of the studycompared the coefficient of fat absorption following oral administrationof the pancrelipase composition in daily doses less than or equal to10,000 lipase units per kilogram of body weight in combinations of5,000, 10,000, 15,000 or 20,000 lipase units per capsule versus aplacebo. The secondary endpoints of the trial evaluated changes in thecoefficient of nitrogen absorption as a determinant of proteinabsorption, cholesterol, fat soluble vitamins, weight, body mass indexand EPI symptoms.

Patients treated with these compositions showed a statisticallysignificant increase in the coefficient of fat absorption andcoefficient of nitrogen absorption as compared to those receiving aplacebo and had fewer symptoms associated with impaired absorption suchas bloating, flatulence, pain and evidence of fat in stools. Increasesin mean cholesterol and vitamin levels were also observed in patientstaking these pancrelipase compositions versus placebo. A statisticallysignificant decrease was obtained in the frequency of stools per day.These compositions were well tolerated by patients, and no drug relatedserious adverse events were observed during the study.

The mean percentage coefficient of fat absorption in patients receivingthese compositions was 88.28% versus 62.76% in patients receivingplacebo. The mean percentage coefficient of nitrogen absorption was87.2% versus 65.7% in patients taking placebo and the mean number ofstools per day decreased in respective patient groups from 2.66 to 1.77.

Example 13

A pediatric, Phase III clinical trial was carried out to evaluate theeffects of treatment with the compositions of Table 34 in an open-labelstudy in 19 CF patients under the age of seven in 11 CF treatmentcenters in the US—the first pancreatic replacement therapy trial of thissize conducted on young children and infants with exocrine pancreaticinsufficiency. The study design involved a seven-day dose stabilizationperiod followed by a seven-day treatment period; patients received 5,000lipase units per capsule daily, with the product being sprinkled on foodas required. The study's primary endpoint was the percentage of“responders,” or those patients without excess fat in stools and withoutsigns and symptoms of malabsorption after one and two weeks oftreatment. Secondary endpoints included weight change, nutritionalstatus, stool frequency and consistency, incidences of bloating, painand flatulence as well as physician and parent or guardian judgment ofclinical symptoms improvement. Product safety was also assessed.

The mean percentage of responders, as defined in the study protocol, atscreening (the beginning of the stabilization period when patients wereon a previous pancreatic enzyme replacement therapy and prior totreatment) was 52.6%. At the end of the stabilization period and the endof the treatment period, the mean percentages of responders were 66.4%and 57.9% respectively. Among the children in the study, malabsorptionsymptoms were significantly lower at the end of the treatment periodthan at screening, consistent with observations regarding control ofmalabsorption symptoms seen in the Phase III trial described in Example12, above. The pancrelipase compositions of the present invention werealso well tolerated by these patients and no drug related seriousadverse events were observed during the trial.

The results showed that the compositions of the present inventioneffectively controlled the signs and symptoms of malabsorption andsupport the results obtained in the pivotal phase III trial described inExample 12. A significant proportion of physicians and patients feltthat the control of symptoms with the compositions of the presentinvention was improved versus previous therapies.

Example 14

A Phase III opened-label, randomized, single center, single treatment,cross-over study was carried out to compare the effects of treatmentwith the pancrelipase compositions of Table 34 to determine thegastrointestinal bioavailability of these compositions in fed conditionsin 10 chronic pancreatitis patients with exocrine pancreaticinsufficiency. Exclusionary drugs (proton pump inhibitors (PPI's),antacids, and drugs capable of altering GI mobility were discontinued 7days prior to entering the study. Patients were randomized to receiveeither Ensure Plus™ (a vitamin fortified nutritional supplementavailable from Abbott) alone or Ensure Plus™ in combination with 75,000USP lipase units (3 capsules containing 20,000 units each plus 3capsules containing 5000 units each) per procedure. The capsules wereopened and their contents mixed with 480 mL of Ensure Plus™ immediatelybefore administration. After a one-day washout period, this procedurewas repeated, except that patients who previously received Ensure Plus™alone received Ensure Plus™ in combination with 75,000 USP lipase units,and patients who previously received the combination of Ensure Plus™ andlipase, received Ensure Plus™ alone. The following day, patientsreceived a physical exam, and blood and urine samples were collected.The bioavailability of the compositions of the present invention wasestimated from the amount of the respective enzymes released andrecovered (i.e., lipase, amylase, and chymotrypsin) in the duodenumfollowing administration of the composition in the presence of EnsurePlus™. Measurements of cholecystokinin levels in the blood, and gastricand duodenal pH were also measured. Lipase activity was measuredaccording to the method of Carriere, F,; Barrowman, J. A.; Verger, R.;Laugier, R. Secretion and contribution to lipolysis of gastric andpancreatic lipases during a test meal in humans. Gastroenterology 1993,105, 876-88. Amylase and chymotrypsin were measured according to themethods described in Carriere, F.; Grandval, P.; Renou, C.; Palomba, A.;Prieri, F.; Giallo, J; Henniges, F.; Sander-Struckmeier, S.; Laugier, R.Quantitative study of digestive enzyme secretion and gastrointestinallipolysis in chronic pancreatitis Clin. Gastroenterol. Hepatol. 2005, 3,28-38.

Treatment with the pancrelipase compositions of the present inventionwas found to result in statistically significant greater amounts ofamylase, lipase and chymotrypsin released in the duodenum of patientsthat received the combination of Ensure Plus™ and pancrelipase, comparedto patients that received Ensure Plus™ only (after correcting for pH asa confounding factor).

The mean bioavailability for lipase, amylase and chymotrypsin for theeight patients who completed the study according to the protocol) was27.5%, 21.6%, and 40.1% respectively. It was found that the patientsfell into two different GI pH subpopulations (“normal pH” and “low pH”).For patients who had “normal pH” values (i.e., patients with a meanduodenal pH greater than 4), the mean bioavailability of lipase, amylaseand chymotrypsin was higher than for the entire study group: 45.6%,26.9%, and 47.7%, respectively. No difference in cholecystokinin valuesbetween the two treatments was observed.

Because the bioavailability for lipase, amylase, and chymotrypsindiffers for “normal pH” and “low pH” patients (particularly for lipase),the efficacy of the pancrelipase compositions or dosage forms of thepresent invention can be enhanced, e.g., in “low pH” patients, byco-administration of medications which increase GI pH, for example PPI'sand antacids. However, the compositions or dosage forms of the presentinvention can be administered without co-administration of e.g., PPI's.

The foregoing description of the invention has been presented for thepurpose of illustration and description. It is not intended to beexhaustive or to limit the invention to the precise form disclosed.Modifications and variations are possible in light of the aboveteachings. The descriptions of the embodiments were chosen in order toexplain and to describe the principles of the present invention and itspractical application, and are not meant to be limiting on the scope ofthe claims.

All publications and patents or patent applications cited herein areincorporated by reference in their entirety to the same extent as ifeach individual publication, patent, or patent application werespecifically and individually indicated incorporated by reference.

1. A method of preparing a pancrelipase composition comprising a plurality of particles each of which is coated with a coating, comprising the steps of: coating particles of pancrelipase in an atmosphere having a moisture content of about 3.6 g water per m³ or less, with a coating comprising an enteric polymer and at least one inorganic material, thereby forming a plurality of delayed release particles having a moisture content of about 3% or less.
 2. The method of claim 1, wherein the atmosphere comprises air, nitrogen, or an inert gas.
 3. The method of claim 1, wherein the particles of pancrelipase are coated with a mixture comprising an enteric polymer dissolved in acetone and at least one inorganic material.
 4. A method of preparing a composition comprising a plurality of particles each of which is coated with a coating, comprising: coating particles of pancrelipase in an atmosphere having a moisture content of about 3.6 g water per m³ or less, with a coating comprising an enteric polymer and at least one inorganic material, thereby forming a plurality of delayed release particles having a water activity of about 0.6 or less.
 5. The method of claim 4, wherein the atmosphere comprises air, nitrogen, or an inert gas.
 6. The method of claim 4, wherein the particles of pancrelipase are coated with a mixture comprising an enteric polymer dissolved in acetone and at least one inorganic material.
 7. A method of preparing a composition comprising a plurality of particles each of which is coated with a coating, comprising: coating particles of a pancrelipase in an atmosphere having a moisture content of about 3.6 g water per m³ or less, with an enteric coating comprising an enteric polymer and at least one inorganic material, thereby forming a plurality of delayed release particles that exhibit a loss of digestive enzyme activity of no more than 15% after six months of accelerated stability testing.
 8. The method of claim 7, wherein the atmosphere comprises air, nitrogen, or an inert gas.
 9. The method of claim 7, wherein the particles of pancrelipase are coated with a mixture comprising an enteric polymer dissolved in acetone and at least one inorganic material. 